While viruses are widely recognized as a cause of infectious disease, certain viruses are also associated with human cancer. The human immune system is central to the control of viral infections and also malignancies shown to be related to oncogenic viruses. Within the complex array of cells, antibodies and immunomodulatory molecules which constitute the human immune system, lymphocytes of thymic origin (T cells) operate in a central role to control viral infections and cancer. Hence, one approach to prevent or treat virus infections and cancer has been to take T-cells from these patients and stimulate and/or expand them in vitro before transfusing them back into the patient. In vivo T cell activation and antigen-specific expansion is generally considered to result from a two signal process wherein the first signal is initiated by the ligation of the T cell receptor/CD3 complex with a major histocompatibility complex class I or class II molecule (MHC Class I or MHC Class II) presenting a peptide antigen. The MHC Class I or MHC Class II and peptide complex is expressed on the surface of a cell (the antigen presenting cell or APC). The peptide antigen originates from a molecule within the cell which undergoes endogenous processing and may be, inter alia, (1) a “self” antigen naturally occurring in the body; (2) a tumour antigen which results from a mutation related to cancer or (3) a viral antigen associated with infection or cancer. The recognition of the antigen by the T cell receptor is considered the first signal and the second signal arises from co-stimulation which results from the ligation of additional surface molecules on the T cell with additional molecules on the APC. The up-regulation and ligation of these co-stimulatory molecules between the T cell and APC may be necessary to effect or enhance T cell activation since the first signal may not be sufficient alone to achieve this. The two signal activation may lead to the expansion of the T cell so that greater numbers of the antigen-specific T cells will be available to control the pathogen or cancer giving rise to the immune response. The canonical understanding of this two signal activation is based on the same APC providing both the first signal and the second signal to the responding T cell such that the co-stimulation is directly associated with antigen recognition. The in vitro activation and expansion of T cells has traditionally been a long, complex and resource intensive process. A typical process may, for example take 8-12 weeks and often employs live “target” virus and/or viral vectors to achieve antigen presentation by antigen presenting cells (APC). Generally T cell activation/expansion requires static conditions rather than stirred or physically agitated culture systems.
One prior art method of culturing antigen specific T cells which recognize the LMP1 and LMP2 antigens of Epstein Barr Virus (EBV) may be summarised as follows:
Preparatory Steps
                In order to achieve the two signal T cell activation and expansion process in a controlled manner, it is useful to create an antigen-presenting cell through transfection of B cells taken from the patient. This is referred to as establishing an autologous lymphoblastoid cell line and is undertaken through infection of the cell with EBV (EBV-LCL). It takes about 6-8 weeks to develop this cell line and thus it is one of the first stages that must be started as part of a culture process which relies upon the EBV-LCL for antigen presentation. It is prepared by culturing B cells from the patient with EBV virus in the presence of cyclosporin A to inhibit EBV-specific T cell outgrowth and elimination of the LCL.                    Prior to the expansion step with the EBV-LCLs, the culture system involves the initial activation and expansion of the LMP-1 and LMP-2 specific T cells with autologous dendritic cells which been transduced with a viral vector Ad5f35-LMP1-LMP2 (encoding the EBV proteins LMP1 and LMP2) (days 0 to 8).            day 9 to 12 the cytolytic T lymphocytes (CTLs) are harvested and re-suspended in fresh medium and re-stimulated with EBV-LCLs transduced with Ad5f35-LMP1-LMP2.            day 13 to 16 cytolytic T lymphocytes are fed with fresh medium and recombinant human IL-2            followed by weekly re-stimulation using CTL:LCL and twice weekly addition of IL-2 for a 4 to 8 week period.                        Dendritic cells for use in the process must also be prepared by taking PBMCs from a patient sample and activating them with IL-4 and GM-CSF to provide adherent PBMCs. These cells are then transduced with a viral vector Ad5f35-LMP1-LMP2 (i.e. encoding the EBV protein LMP1 and LMP2). Finally the dendritic cells are matured by the addition of IL-1β, IL-6, PGE-1 and TNF-α.Summary of T Cell Expansion Steps(Also Referred to as Preparation of Cytotoxic T Lymphocytes (CTLs))        Once prepared the transduced dendritic cells are cultured with fresh PBMCs from the patient for a period of about 10 days.        The T cells obtained from this step are then cultured with the transduced EBV-LCLs for a period of about 1 week.        Then the T cells obtained from the latter step are then cultured with transduced EBV-LCLs in the presence of IL-2 for a further 10 days to provide an autologous T cell antigen specific product        This process is repeated until sufficient T cells have expanded.        
J Immunother Vol 33, Number 3, April 2010 describes a faster and more efficient way of culturing the cells over a period of 23 days employing a system from Wilson Wolf known as the GRex™ system. However, this rapid process still employs the traditional viral stimuli for the cells.
There are various problems with the prior art strategy:    (i) the use of live virus such as EBV and viral vectors has the potential to cause an immunodominant response against the vector which may interfere with efficient generation of target virus specific CTL's;    (ii) the use of live virus is an impediment to progression to phase 3 trials due to safety concerns;    (iii) the requirement for B cells to manufacture the EBV-LCL, now that Rituxan (which depletes B cells) has become standard therapy for most lymphoma patients means the technique cannot be employed for many patients;    (iv) the duration of manufacturing (a minimum of 6 weeks to establish the EBV-LCL and another 5 to 7 weeks for CTL expansion) is very inconvenient, impractical and economically challenging;    (v) the complexity of cell manipulation provides many opportunities for error and contamination of the product, hence, the principles of good manufacturing practise (GMP) are difficult to comply with, and    (vi) the autologous antigen presenting cells used to stimulate the T cell expansion can express antigens other than the target antigen, which may reduce the purity of the antigen-specific T cells which are desired for the therapeutic T cell product.
Nevertheless skilled persons have been reluctant to move away from the established processes because each step was thought necessary to generate a product with therapeutic characteristics and in particular to generate T cell populations that are suitable for recognising cells infected by live viruses and cancers expressing viral antigens, in vivo.
The present disclosure provides a method for the rapid and efficient production of antigen specific T cells with specificity to a target antigen.